Chronic liver disease is responsible for over 1.4 million disability adjusted life years annually and ranks in the United States among the top 7 disease-related causes of death between the age of 25 and 64 years. For end-stage liver failure, orthotopic liver transplantation remains the current treatment of choice. However, patients suffering from acute or acute-on-chronic liver failure may benefit from temporary extracorporeal artificial liver support used to tide them over until transplantation or to allow regeneration of their own liver to occur.
Bioartificial liver (BAL) systems seem to be a promising solution for this purpose. These systems allow for extracorporeal blood (plasma) treatment relying on functional liver cell cultures for detoxification and synthetic function. Over the last 2 decades, many BAL systems have been devised, only some of which systems have been applied in a clinical (pilot) setting. BALs are based on a bioreactor facilitating functional liver cells and can be employed to compensate for the loss of hepatocellular function by perfusing the reactor with the subject's blood plasma. Some BAL systems comprise a bioreactor in combination with a non-biological detoxification modality, such as a charcoal column or a bilirubin column (so called hybrid systems). For a more complete understanding of existing BAL technology, Park and Lee1 or Sgroi et al.2 may be referred to, providing comprehensive overviews of the different types of reactors and the different system configurations developed by various groups up to 2009.
The cell source employed in the BAL bioreactor should exhibit high hepatic functionality. Although the pathogenesis of hepatic encephalopathy (HE), one of the major causes of death of ALF, is multifactorial, it is generally accepted that hyperammonemia plays a crucial role. Increased plasma ammonia levels are toxic for the brain, contribute to brain edema and stimulate inhibitory neurotransmission. A very essential liver function is detoxifying blood ammonia, primarily by urea synthesis and secondarily by glutamine synthesis. Urea is non toxic and is rapidly excreted by the kidneys. Glutamine is non-toxic as well and can be used as metabolic substrate by different organs among which the intestine. In addition, the cell source should display detoxification of other accumulating endogenous toxic compounds mainly through the cytochrome P450 system, synthesis of blood proteins and homeostasis of amino acids, lipids and carbohydrates.
Many liver cell types have been used for preclinical and clinical application of BAL devices. The choice for the optimal cell source for BAL, support devices is, however, still a matter of debate. Particularly, the availability, degree of liver specific function, and safety aspects are ongoing issues. Primary hepatocytes, either allogeneic or xenogeneic, have an excellent function and from that perspective would appear to be the favorite cell sources for BAL application.
However, mature porcine hepatocytes are not attractive, for clinical application because of the obvious risks and objections related to methods of treatment of human beings involving xenotransplantation.
Mature human hepatocytes, on the other hand, are scarce, as their sources are limited to either discarded donor livers or small parts obtained during liver resections.
Human hepatic cell lines have the advantage of an infinite proliferation capacity and can potentially serve as a stable cell source. In addition, the use of such cell lines from human origin would effectively avoid the xenotransplantation associated risks and objections. From these perspectives human hepatocyte cell lines seem to offer the most promising starting point for successful BAL development. Not surprisingly therefore, a lot of effort has been invested in finding and developing human hepatocyte cell lines having the required hepatic functionalities. So far, these efforts have only resulted in very limited success though.
The potential of a human fetal liver cell line cBAL111 for application in BAL systems has been tested and described by Poyck et al.3. Poyck et al. reported that cBAL111 eliminated ammonia at a rate up to 49% of that in primary porcine hepatocytes (PPH), despite a low urea production. Their synthetic functions (albumin production: 6%) and detoxification functions (lidocaine elimination: 1%) were to be low.
Uses of HepG2 cells or genetically modified variants thereof, in several BAL systems have been described by e.g. Nyberg et al.4, Wang et al.5, Enosawa et al.6, and Takahashi et al.7. It is now well-established that the urea cycle is not maintained in HepG2 cells resulting in the lack of ammonia detoxification via this route. Gene expression data from HepG2 cells reveals limited expression of three urea cycle genes Carbamoyl Phosphate Synthase I (CPS), Arginosuccinate Synthetase (ASS) and Arginosuccinate Lyase, whereas no expression of Ornithine Transcarbamylase can be established.
According to Mavri-Damelin et al.8, a HepG2 sub-clone, designated C3A, nevertheless had been found to produce urea. Their research confirmed that gene expression of ornithine transcarbamylase (OTC) and arginase I (Arg I), were completely absent. Arginase II (Arg II) mRNA and protein was expressed at high levels in C3A cells though and was inhibited by Nω-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The authors conclude that the urea cycle is non-functional in C3A cells, and that these cells therefore cannot provide ammonia detoxification in a BAL system via urea. The authors note that this emphasizes the continued requirement for developing a component capable of a full repertoire of liver function, including an intact urea cycle to detoxify ammonia.
Kosuge et al.9 reported that in gene expression profiles of bioreactor grown FLC-4, FLC-5 and FLC-7 cells some genes for liver functions were expressed at a level similar to that in normal liver, although none of the cell lines expressed the complete set of genes encoding ammonium metabolising enzymes or cytochrome P450 species. The use of a BAL system loaded with FLC-4 cells for treating pigs with hepatic dysfunction was reported by Kanai et al.10. However, the FL-4 cells were defect in their urea cycle.
Saito et al.11 reported the development of a BAL system comprising FLC-5 cells. To assess hepatocyte function of the cells incubated in the BAL, expression of urea cycle and albumin synthesis enzymes were studied. The authors reported the presence of urea in the medium and an increase in the expression of urea cycle enzymes ASS and Arg I for the BAL incubated cells, whereas albumin synthesis had decreased. All in all, no FLC cell line has been obtained so far providing complete metabolic functionality that would render it suitable for clinical BAL application.
US 2005/0064594 discloses a liver cell line, designated HepaRG, which approximates human hepatocytes in cytochrome P450 3A activity (CYP3A4), which is a key factor in detoxification, after culturing for 14 days in HepaRG medium with 2% of dimethylsulfoxide (DMSO) after an initial 14-days proliferation phase without the addition of DMSO. In these cultures growth as well as many of the required hepatic functions other than CYP3A4, like urea synthesis and albumin synthesis are suppressed.
In summary, each of the human cell lines described above all have certain detoxification and/or metabolic liver functionalities, whereas certain other, essential, detoxification and/or metabolic liver functionalities are substantially reduced compared to those in freshly isolated human hepatocytes. In particular, until now, it has been problematic to acquire cells with a broad spectrum functionality, resembling that of freshly isolated human hepatocytes, to the extent that they are in fact suitable for successful clinical BAL application. Consequently, so far, it has proven impossible to develop a human cell line based BAL system providing complete hepatic functionality, especially a system providing both detoxification functionality as well as complete metabolic functionality.
It was the objective of the present inventors to provide adequate solutions to these short-comings of the prior art cells and BAL systems, thereby bringing successful clinical application a significant step closer.